C-terminal antibody variants

ABSTRACT

The invention generally relates to anti-sclerostin antibodies having C-terminal modifications, and compositions comprising such antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority to U.S.Provisional Application No. 62/650,762, filed Mar. 30, 2018 and U.S.Provisional Application No. 62/812,741, filed Mar. 1, 2019, thedisclosures of which are incorporated herein by reference in theirentireties.

TECHNICAL FIELD OF THE INVENTION

The invention generally relates to anti-sclerostin antibodies having atleast one C-terminal modification and compositions comprising suchantibodies.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: ASCII (text) file named“52080_SeqListing.txt,” 21,006 bytes, created on Mar. 21, 2019.

INCORPORATION BY REFERENCE

The following applications are hereby incorporated by reference in theirentirety: International Patent Publication No. PCT/US2012/049331, filedAug. 2, 2012, which claims priority to U.S. Provisional PatentApplication No. 61/515,191, filed Aug. 4, 2011; U.S. patent applicationSer. No. 11/410,540, filed Apr. 25, 2006, which claims priority to U.S.Provisional Patent Application No. 60/792,645, filed Apr. 17, 2006, U.S.Provisional Patent Application No. 60/782,244, filed Mar. 13, 2006, U.S.Provisional Patent Application No. 60/776,847, filed Feb. 24, 2006, andU.S. Provisional Patent Application No. 60/677,583, filed May 3, 2005;and U.S. patent application Ser. No. 11/411,003 (issued as U.S. Pat. No.7,592,429), filed Apr. 25, 2006, which claims priority to U.S.Provisional Patent Application No. 60/792,645, filed Apr. 17, 2006, U.S.Provisional Patent Application No. 60/782,244, filed Mar. 13, 2006, U.S.Provisional Patent Application No. 60/776,847, filed Feb. 24, 2006, andU.S. Provisional Patent Application No. 60/677,583, filed May 3, 2005.The following applications also are hereby incorporated by reference:U.S. patent application Ser. No. 12/212,327, filed Sep. 17, 2008, whichclaims priority to U.S. Provisional Patent Application No. 60/973,024,filed Sep. 17, 2007; and U.S. patent application Ser. No. 12/811,171,filed Jun. 29, 2010, which is a U.S. National Phase Application pursuantto 35 U.S.C. § 371 of International Patent Application No.PCT/US08/86864, filed on Dec. 15, 2008, which claims priority to U.S.Provisional Patent Application No. 61/013,917, filed Dec. 14, 2007.

BACKGROUND

Loss of bone mineral content can be caused by a wide variety ofconditions and may result in significant medical problems. For example,osteoporosis is a debilitating disease in humans and is characterized bymarked decreases in skeletal bone mass and mineral density, structuraldeterioration of bone, including degradation of bone microarchitectureand corresponding increases in bone fragility (i.e., decreases in bonestrength), and susceptibility to fracture in afflicted individuals.Although osteoporosis has been regarded as an increase in the risk offracture due to decreased bone mass, few of the presently availabletreatments for skeletal disorders can increase the bone density ofadults, and most of the presently available treatments work primarily byinhibiting further bone resorption rather than stimulating new boneformation. Estrogen is now being prescribed to retard bone loss.However, some controversy exists over whether patients gain anylong-term benefit and whether estrogen has any effect on patients over75 years old. Moreover, use of estrogen is believed to increase the riskof breast and endometrial cancer. Calcitonin, osteocalcin with vitaminK, or high doses of dietary calcium, with or without vitamin D, havealso been suggested for postmenopausal women. High doses of calcium,however, often have undesired gastrointestinal side effects, and serumand urinary calcium levels must be continuously monitored (e.g., Khoslaand Riggs, Mayo Clin. Proc. 70:978982, 1995). Other current therapeuticapproaches to osteoporosis include bisphosphonates (e.g., Fosamax™Actonel™, Bonviva™, Zometa™, olpadronate, neridronate, skelid, bonefos),parathyroid hormone, calcilytics, calcimimetics (e.g., cinacalcet),statins, anabolic steroids, lanthanum and strontium salts, and sodiumfluoride. Such therapeutics, however, are often associated withundesirable side effects (see Khosla and Riggs, supra).

Sclerostin, the product of the SOST gene, is absent in sclerosteosis, askeletal disease characterized by bone overgrowth and strong dense bones(Brunkow et al., Am. J. Hum. Genet., 68:577-589, 2001; Balemans et al.,Hum. Mol. Genet., 10:537-543, 2001). The amino acid sequence of humansclerostin is reported by Brunkow et al. ibid and is disclosed herein asSEQ ID NO:1. Sclerostin is valuable target for mediating increases inbone density.

SUMMARY

In one aspect, described herein is an antibody that specifically bindsto sclerostin of SEQ ID NO: 1 and comprises a set of six CDRs set forthin SEQ ID NOs: 2-7, wherein the antibody comprises a heavy chaincomprising the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at theC-terminus of the heavy chain. In some embodiments, the antibodycomprises a light chain variable region comprising an amino acidsequence set forth in SEQ ID NO: 9 and a heavy chain variable regioncomprising an amino acid sequence set forth in SEQ ID NO: 10. In someembodiments, the antibody comprises the amino acid sequencePro-Ala-Arg-Gly-Lys (SEQ ID NO: 11) at the C-terminus of the heavychain. In some embodiments, the antibody comprises a first heavy chaincomprising the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at theC-terminus of the first heavy chain and a second heavy chain comprisinga wild-type heavy chain amino acid sequence (i.e., lacking theC-terminal Pro-Ala-Arg-Gly). In some embodiments, the antibody comprisesthe light chain amino acid sequence set forth in SEQ ID NO: 12 and theheavy chain amino acid sequence set forth in SEQ ID NO: 13. In someembodiments, the antibody comprises the amino acid sequencePro-Ala-Arg-Gly-Lys (SEQ ID NO: 11) at the C-terminus of the heavychain. In some embodiments, the antibody comprises the light chain aminoacid sequence set forth in SEQ ID NO: 12 and the heavy chain amino acidsequence set forth in SEQ ID NO: 14.

In some embodiments, the C-terminus of one of the heavy chains of theantibody is amidated (i.e., the antibody is singly amidated). In someembodiments, the C-terminus of both heavy chains of the antibody isamidated (i.e., the antibody is double amidated).

Pharmaceutical compositions comprising a population of the antibodiesdescribed herein and a pharmaceutically acceptable carrier are alsoprovided by the disclosure. In some embodiments, the pharmaceuticalcomposition comprises a mixture of antibodies that specifically bind tosclerostin of SEQ ID NO: 1, wherein the mixture of antibodies comprisesa population of antibodies comprising a heavy chain having the aminoacid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of theheavy chain and a pharmaceutically acceptable carrier. In someembodiments, about 3-5% of the antibodies in the composition are apopulation of the antibodies comprising a heavy chain having the aminoacid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of theheavy chain. In some aspects, less than 70% of the population ofantibodies are amidated on one or both heavy chains. In some aspects,all or part of the population of antibodies comprise a single heavychain comprising a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence,which is optionally amidated. In some aspects, all or part of thepopulation of antibodies comprise a C-terminal Pro-Ala-Arg-Gly (SEQ IDNO: 8) sequence in both heavy chains, and both heavy chains areoptionally amidated. Optionally, less than about 35% of the populationof antibodies is singly amidated and/or less than about 35% of thepopulation of antibodies are amidated on both heavy chains and/or lessthan about 35% of the population of antibodies comprise heavy chainsthat are not amidated. In this regard, in various aspects, about 33% ofthe population of antibodies are not amidated, about 33% of thepopulation of antibodies comprise one amidated heavy chain, and about33% of the population of antibodies comprise two amidated heavy chains.

In some embodiments, the composition further comprises a calcium salt,an acetate buffer, a polyol and a surfactant. In some embodiments, theacetate salt comprises calcium acetate, the acetate buffer comprisessodium acetate, the polyol comprises sucrose and the surfactantcomprises polysorbate 20. In some embodiments, the composition comprises55 mM acetate, 13 mm calcium, 6.0% (w/v) sucrose, and 0.006% (w/v)polysorbate 20, pH 5.2.

Also provided by the disclosure is a method of increasing bone mineraldensity in a subject in need thereof comprising administering thecomposition described herein to the subject in an amount effective toincrease bone mineral density in the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a nucleic acid sequence which encodes a portion of theC-terminus of wild-type romosozumab.

FIG. 2 provides a nucleic acid sequence which encodes a portion of theC-terminus of the romosozumab C-terminal variant (PARG variant).

FIG. 3 is a graph showing the zoomed in UV profile of wild-typeromosozumab (dotted line) overlaid with and the romosozumab PARG variant(solid line) that has been digested by Lys-C and analyzed by LC/MSpeptide mapping.

FIG. 4 is a graph showing the cation exchange (CEX) profile ofcarboxypeptidase treated romosozumab PARG variant (dotted line) overlaidwith untreated romosozumab PARG variant (solid line).

FIG. 5 is a graph showing the percent recovery from the Scissor model ofa subcutaneous injection shown as a function of time. Wild typeromosozumab (circles) and PARG c-terminal variant romosozumab diffuse atdifferent rates for the simulated injection site.

FIG. 6 is a graph showing that both wild type romosozumab and PARGC-terminal variant romosozumab bound FcRn similarly and that FcRnbinding was not affected by the PARG mutation.

FIG. 7 is a graph showing that the relative binding of PARG C-terminalvariant romosozumab to FcγRIIa (131H) was much higher than wild-typeromosozumab.

DETAILED DESCRIPTION

The disclosure provides an antibody that specifically binds tosclerostin, wherein the antibody comprises a heavy chain comprising asequence of amino acids comprising Pro-Ala-Arg-Gly (SEQ ID NO: 8) at theC-terminus of the heavy chain. In some embodiments, the antibodycomprises a first heavy comprising a sequence of amino acids comprisingPro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of the heavy chain anda second heavy chain comprising a wild-type heavy chain amino acidsequence. In some embodiments, the antibody comprises a sequence ofamino acids comprising Pro-Ala-Arg-Gly-Lys (SEQ ID NO: 11) at theC-terminus of the heavy chain. Pharmaceutical compositions comprisingthe antibody (or mixture of antibodies) and methods of using theantibody are also provided.

An “anti-sclerostin antibody” or an “antibody that binds to sclerostin”is an antibody that binds to sclerostin of SEQ ID NO: 1 or portionsthereof. Recombinant human sclerostin/SOST is commercially availablefrom, e.g., R&D Systems (Minneapolis, Minn., USA; 2006 Catalog#1406-ST-025). U.S. Pat. Nos. 6,395,511 and 6,803,453, and U.S. PatentPublication Nos. 2004/0009535 and 2005/0106683 refer to anti-sclerostinantibodies generally. Examples of sclerostin antibodies suitable for usein the context of the invention also are described in U.S. PatentPublication Nos. 2007/0110747 and 2007/0072797, which are herebyincorporated by reference. Additional information regarding materialsand methods for generating sclerostin antibodies can be found in U.S.Patent Publication No. 20040158045 (hereby incorporated by reference).

The term “antibody” refers to an intact immunoglobulin molecule(including polyclonal, monoclonal, chimeric, humanized, and/or humanversions having full length heavy and/or light chains).

“Specifically binds” as used herein means that the antibodypreferentially binds the antigen over other proteins. In someembodiments, “specifically binds” means the antibody has a higheraffinity for the antigen than for other proteins. Antibodies thatspecifically bind an antigen may have a binding affinity for the antigenof less than or equal to 1×10⁻⁷ M, less than or equal to 2×10⁻⁷ M, lessthan or equal to 3×10⁻⁷ M, less than or equal to 4×10⁻⁷ M, less than orequal to 5×10⁻⁷ M, less than or equal to 6×10⁻⁷ M, less than or equal to7×10⁻⁷ M, less than or equal to 8×10⁻⁷ M, less than or equal to 9×10⁻⁷M, less than or equal to 1×10⁻⁸ M, less than or equal to 2×10⁻⁸ M, lessthan or equal to 3×10⁻⁸ M, less than or equal to 4×10⁻⁸ M, less than orequal to 5×10⁻⁸ M, less than or equal to 6×10⁻⁸ M, less than or equal to7×10⁻⁸ M, less than or equal to 8×10⁻⁸ M, less than or equal to 9×10⁻⁸M, less than or equal to 1×10⁻⁹ M, less than or equal to 2×10⁻⁹ M, lessthan or equal to 3×10⁻⁹ M, less than or equal to 4×10⁻⁹ M, less than orequal to 5×10⁻⁹ M, less than or equal to 6×10⁻⁹ M, less than or equal to7×10⁻⁹ M, less than or equal to 8×10⁻⁹ M, less than or equal to 9×10⁻⁹M, less than or equal to 1×10⁻¹⁰ M, less than or equal to 2×10⁻¹⁰ M,less than or equal to 3×10⁻¹⁰ M, less than or equal to 4×10⁻¹⁰ M, lessthan or equal to 5×10⁻¹⁰ M, less than or equal to 6×10⁻¹⁰ M, less thanor equal to 7×10⁻¹⁰ M, less than or equal to 8×10⁻¹⁰ M, less than orequal to 9×10⁻¹⁰ M, less than or equal to 1×10⁻¹¹ M, less than or equalto 2×10⁻¹¹ M, less than or equal to 3×10⁻¹¹ M, less than or equal to4×10⁻¹¹ M, less than or equal to 5×10⁻¹¹ M, less than or equal to6×10⁻¹¹ M, less than or equal to 7×10⁻¹¹ M, less than or equal to8×10⁻¹¹ M, less than or equal to 9×10⁻¹¹ M, less than or equal to1×10⁻¹² M, less than or equal to 2×10⁻¹² M, less than or equal to3×10⁻¹² M, less than or equal to 4×10⁻¹² M, less than or equal to5×10⁻¹² M, less than or equal to 6×10⁻¹² M, less than or equal to7×10⁻¹² M, less than or equal to 8×10⁻¹² M, or less than or equal to9×10⁻¹² M.

In some or any embodiments, the antibody binds to sclerostin of SEQ IDNO: 1, or a naturally occurring variant thereof, with an affinity (Kd)of less than or equal to 1×10⁻⁷ M, less than or equal to 1×10⁻⁸M, lessthan or equal to 1×10⁻⁹ M, less than or equal to 1×10⁻¹⁰ M, less than orequal to 1×10⁻¹¹ M, or less than or equal to 1×10⁻¹² M. Affinity isdetermined using a variety of techniques, an example of which is anaffinity ELISA assay. In various embodiments, affinity is determined bya BIAcore assay. In various embodiments, affinity is determined by akinetic method. In various embodiments, affinity is determined by anequilibrium/solution method. U.S. Patent Publication No. 2007/0110747(the disclosure of which is incorporated herein by reference) containsadditional description of affinity assays suitable for determining theaffinity (Kd) of an antibody for sclerostin.

In some or any embodiments, the antibody (or antibody fragments thereof)binds to a sclerostin polypeptide comprising the amino acid sequence setforth in SEQ ID NO: 1 and binds a region of sclerostin comprising thesequence of SEQ ID NO: 5 (CGPARLLPNAIGRGKWWRPSGPDFRC; corresponding toamino acids 86-111 of SEQ ID NO: 1). This region is also referred toherein as the “loop 2” region of sclerostin. Regions of sclerostinoutside of the loop 2 region are defined herein as “non-loop 2 regions.”Alternatively or in addition, the anti-sclerostin antibody binds to asclerostin polypeptide comprising amino acids 57-146 of SEQ ID NO: 1.Alternatively or in addition, the anti-sclerostin antibody binds to asclerostin polypeptide comprising amino acids 89-103 of SEQ ID NO: 1and/or amino acids 137-151 of SEQ ID NO: 1. In some or any embodiments,the sclerostin polypeptide that is a fragment of full length sclerostinretains the tertiary structure of the corresponding polypeptide regionof human sclerostin of SEQ ID NO: 1.

In some or any embodiments, the anti-sclerostin antibody describedherein preferably modulates sclerostin function in the cell-based assaydescribed in U.S. Patent Publication No. 2007/0110747 and/or the in vivoassay described in U.S. Patent Publication No. 20070110747 and/or bindto one or more of the epitopes described in U.S. Patent Publication No.2007/0110747 and/or cross-block the binding of one of the antibodiesdescribed in U.S. Patent Publication No. 2007/0110747 and/or arecross-blocked from binding sclerostin by one of the antibodies describedin U.S. Patent Publication No. 2007/0110747 (incorporated by referencein its entirety and for its description of assays for characterizing ananti-sclerostin antibody).

“CDR” refers to the complementarity determining region within antibodyvariable sequences. There are three CDRs in each of the variable regionsof the heavy chain and the light chain, which are designated CDR1, CDR2and CDR3, for each of the variable regions. The term “set of six CDRs”as used herein refers to a group of three CDRs that occur in the lightchain variable region and heavy chain variable region, which are capableof binding the antigen. The exact boundaries of CDRs have been defineddifferently according to different systems. The system described byKabat (Kabat et al., Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987) and (1991)) notonly provides an unambiguous residue numbering system applicable to anyvariable region of an antibody, but also provides precise residueboundaries defining the three CDRs. These CDRs may be referred to asKabat CDRs. Chothia and coworkers (Chothia & Lesk, J. Mol. Biol.196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) foundthat certain sub-portions within Kabat CDRs adopt nearly identicalpeptide backbone conformations, despite having great diversity at thelevel of amino acid sequence. These sub-portions were designated as L1,L2 and L3 or H1, H2 and H3 where the “L” and the “H” designates thelight chain and the heavy chains regions, respectively. These regionsmay be referred to as Chothia CDRs, which have boundaries that overlapwith Kabat CDRs. Other boundaries defining CDRs overlapping with theKabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) andMacCallum (J Mol Biol 262(5):73245 (1996)). Still other CDR boundarydefinitions may not strictly follow one of the above systems, but willnonetheless overlap with the Kabat CDRs, although they may be shortenedor lengthened in light of prediction or experimental findings thatparticular residues or groups of residues or even entire CDRs do notsignificantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, althoughpreferred embodiments use Kabat or Chothia defined CDRs.

CDRs are obtained by, e.g., constructing polynucleotides that encode theCDR of interest. Such polynucleotides are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionusing mRNA of antibody-producing cells as a template (see, for example,Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106(1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,”in Monoclonal Antibodies Production, Engineering and ClinicalApplication, Ritter et al. (eds.), page 166, Cambridge University Press(1995); and Ward et al., “Genetic Manipulation and Expression ofAntibodies,” in Monoclonal Antibodies: Principles and Applications,Birch et al., (eds.), page 137, Wiley-Liss, Inc. (1995)).

In various aspects, the antibody comprises at least one CDR sequencehaving at least 75% identity (e.g., at least 75%, 80%, 85%, 90%, 95% or100% identity) to a CDR selected from CDR-H1, CDR-H2, CDR-H3, CDR-L1,CDR-L2, and CDR-L3 wherein CDR-H1 has the sequence given in SEQ ID NO:2, CDR-H2 has the sequence given in SEQ ID NO: 3, CDR-H3 has thesequence given in SEQ ID NO: 4, CDR-L1 has the sequence given in SEQ IDNO: 5, CDR-L2 has the sequence given in SEQ ID NO: 6 and CDR-L3 has thesequence given in SEQ ID NO: 7. The anti-sclerostin antibody, in variousaspects, comprises two of the CDRs or six of the CDRs.

In a preferred embodiment, the anti-sclerostin antibody comprise a setof six CDRs as follows: CDR-H1 of SEQ ID NO: 2, CDR-H2 of SEQ ID NO: 3,CDR-H3 of SEQ ID NO: 4, CDR-L1 of SEQ ID NO: 5, CDR-L2 of SEQ ID NO: 6and CDR-L3 of SEQ ID NO: 7.

In some or any embodiments, the antibody comprises a light chainvariable region comprising an amino acid sequence having at least 75%identity (e.g., at least 75%, 80%, 85%, 90%, 95% or 100% identity) tothe amino acid sequence set forth in SEQ ID NO: 9 and a heavy chainvariable region comprising an amino acid sequence having at least 75%identity (e.g., at least 75%, 80%, 85%, 90%, 95% or 100% identity) tothe amino acid sequence set forth in SEQ ID NO: 10. In various aspects,the difference in the sequence compared to SEQ ID NO: 9 or 10 liesoutside the CDR region in the corresponding sequences. In some or anyembodiments, the antibody comprises a light chain variable regioncomprising an amino acid sequence set forth in SEQ ID NO: 9 and a heavychain variable region comprising an amino acid sequence set forth in SEQID NO: 10.

In some or any embodiments the anti-sclerostin antibody comprises all orpart of a heavy chain (e.g., two heavy chains) comprising an amino acidsequence having at least 75% identity (e.g., at least 75%, 80%, 85%,90%, 95% or 100% identity) to the amino acid sequence set forth in SEQID NO: 16 and all or part of a light chain (e.g., two light chains)comprising an amino acid sequence having at least 75% identity (e.g., atleast 75%, 80%, 85%, 90%, 95% or 100% identity) to the amino acidsequence set forth in SEQ ID NO 12.

The antibody comprises a heavy chain comprising the amino acid sequencePro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of the heavy chains. Insome embodiments, the C-terminus of both heavy chains of the antibodycomprises the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8). Insome embodiments, the antibody comprises a first heavy chain comprisingthe amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) and a secondheavy chain comprising a wild-type amino acid sequence. The antibody, invarious aspects, comprises the light chain amino acid sequence set forthin SEQ ID NO: 12 and the heavy chain amino acid sequence set forth inSEQ ID NO: 13.

Alternatively, in some or any embodiments, the antibody comprises asequence of amino acids comprising Pro-Ala-Arg-Gly-Lys (SEQ ID NO: 11)at the C-terminus of a heavy chain, optionally at the C-terminus of bothheavy chains. In some embodiments, the antibody comprises a first heavychain comprising the amino acid sequence Pro-Ala-Arg-Gly-Lys (SEQ ID NO:11) and a second heavy chain comprising a wild-type amino acid sequence(i.e., without the C-terminal Pro-Ala-Arg-Gly-Lys (SEQ ID NO: 11)). Theantibody, in various aspects, comprises the light chain amino acidsequence set forth in SEQ ID NO: 12 and the heavy chain amino acidsequence set forth in SEQ ID NO: 14.

Examples of other anti-sclerostin antibodies include, but are notlimited to, the anti-sclerostin antibodies disclosed in InternationalPatent Publication Nos. WO 2008/092894, WO 2008/115732, WO 2009/056634,WO 2009/047356, WO 2010/100200, WO 2010/100179, WO 2010/115932, and WO2010/130830 (each of which is incorporated by reference herein in itsentirety).

It will be understood by one skilled in the art that some proteins, suchas antibodies, may undergo a variety of posttranslational modifications.The type and extent of these modifications often depends on the hostcell line used to express the protein as well as the culture conditions.Such modifications may include variations in glycosylation, methionineoxidation, diketopiperizine formation, aspartate isomerization andasparagine deamidation. A frequent modification is the loss of acarboxy-terminal basic residue (such as lysine or arginine) due to theaction of carboxypeptidases (as described in Harris, R J. Journal ofChromatography 705:129-134, 1995).

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983],entirely incorporated by reference), acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

In some or any embodiments, the C-terminus of the heavy chain of theantibody, comprising the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO:8), is amidated. In some or any embodiments, both heavy chains of theantibody comprise the amino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8)and both heavy chains are amidated. In some embodiments, the glycine isamidated. Amidation can occur, e.g., as described in Prigg, S. T. etal., “New insights into copper monooxygenases and peptide amidation:structure, mechanism and function”, Cell. Mol. Life Sci. 57 (2000)1236-1259. The enzyme peptidylglycine α-amidating monooxygenase (PAM)can catalyze the amidation of glycine. PAM has two active domains,peptidylglycine α-hydroxylating monooxygenase (PHM) andpeptidyl-α-hydroxylglycine α-amidating lyase (PAL). PHM catalyzes theconversion of peptidylglycine (along with ascorbate and oxygen) topeptidyl α-hydroxylglycine (along with semidehydrogenascorbate andwater). In turn, PAL catalyzes the conversion of peptidylα-hydroxylglycine to an amidated peptide (and glyoxylate).

Amidation of an antibody can be controlled by altering certainconditions during the cell culture process. For example, copper (e.g.,in ferric ammonium citrate) and/or oxygen levels may be used toinfluence amidation levels. It is contemplated that increasing copperconcentration (e.g., in the media) or oxygen availability (e.g., duringculturing) may increase amidation by impacting the activity of an enzymesuch as PHM.

Pharmaceutical Compositions

The disclosure provides a pharmaceutical composition comprising apopulation of the antibody described herein together with apharmaceutically effective diluent, carrier, solubilizer, emulsifier,preservative, and/or adjuvant. Pharmaceutical compositions of theinvention include, but are not limited to, liquid, frozen, andlyophilized compositions.

The disclosure also provides a pharmaceutical composition comprising amixture of antibodies that specifically bind to sclerostin of SEQ ID NO:1 and a pharmaceutically acceptable carrier, wherein about 3-5% of theantibodies in the composition are a population of antibodies describedherein (e.g., antibodies comprising set of six CDRs set forth in SEQ IDNOs: 2-7 and having a heavy chain (or two heavy chains) comprising theamino acid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus ofthe heavy chain(s)). The disclosure also contemplates compositionscomprising alternative amounts (e.g., 5-10%, 1-3%, 3-15%, 2-10%, 4-20%,1-5%) of the population of antibodies described herein (e.g., antibodiescomprising set of six CDRs set forth in SEQ ID NOs: 2-7 and having aheavy chain (or two heavy chains) comprising the amino acid sequencePro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of the heavy chain(s)).

In some embodiments, less than 70% of the antibodies of the population(e.g., about 69%, about 68%, about 67%, about 66%, about 65%, about 64%,about 63%, about 62%, about 61%, about 60%, about 59%, about 58%, about57%, about 56%, about 55%, about 54%, about 53%, about 52%, about 51%,about 50%, about 49%, about 48%, about 47%, about 46%, about 45%, about44%, about 43%, about 42%, about 41%, about 40%, about 39%, about 38%,about 37%, about 36%, about 35%, about 34%, about 33%, about 32%, about31%, about 30%, about 29%, about 28%, about 27%, about 26%, about 25%,about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about18%, about 17%, about 16%, about 15%, about 14%, bout 13%, about 12%,about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%,about 4%, about 3%, about 2%, about 1% or less) comprise a heavy chaincomprising a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence, whichis optionally amidated. In some embodiments, less than 35% (e.g., about34%, about 33%, about 32%, about 31%, about 30%, about 29%, about 28%,about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%,about 14%, bout 13%, about 12%, about 11%, about 10%, about 9%, about8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%or less) of the antibodies of the population comprise a C-terminalPro-Ala-Arg-Gly (SEQ ID NO: 8) sequence on both heavy chains, where bothheavy chains are optionally amidated. It is also contemplated that bothheavy chains comprise the C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8)sequence but only one of the chains is amidated. In some embodiments,less than 35% (e.g., about 34%, about 33%, about 32%, about 31%, about30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%,about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about17%, about 16%, about 15%, about 14%, bout 13%, about 12%, about 11%,about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%,about 3%, about 2%, about 1% or less) of the antibodies in thecomposition comprise a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8)sequence that is not amidated. In some embodiments, about 33% ofantibodies of the population comprise a C-terminal Pro-Ala-Arg-Gly (SEQID NO: 8) sequence that is amidated, about 33% of the antibodies of thepopulation comprise C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequenceson both heavy chains which are both amidated, and about 33% of theantibodies of the population comprise heavy chain(s) with a C-terminalPro-Ala-Arg-Gly (SEQ ID NO: 8) sequence but which are not amidated.

In some embodiments, the pharmaceutical composition contains formulationmaterials for modifying, maintaining or preserving, for example, the pH,osmolarity, viscosity, clarity, color, isotonicity, odor, sterility,stability, rate of dissolution or release, adsorption or penetration ofthe composition. In such embodiments, suitable formulation materialsinclude, but are not limited to, amino acids (such as glycine,glutamine, asparagine, arginine, proline, or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. See,REMINGTON'S PHARMACEUTICAL SCIENCES, 18″ Edition, (A. R. Genrmo, ed.),1990, Mack Publishing Company.

Selection of the particular formulation materials described herein maybe driven by, for example, the intended route of administration,delivery format and desired dosage. See, for example, REMINGTON'SPHARMACEUTICAL SCIENCES, supra. The primary vehicle or carrier in apharmaceutical composition may be either aqueous or non-aqueous innature. For example, a suitable vehicle or carrier may be water forinjection, physiological saline solution or artificial cerebrospinalfluid, possibly supplemented with other materials common in compositionsfor parenteral administration. Neutral buffered saline or saline mixedwith serum albumin are further exemplary vehicles. In specificembodiments, pharmaceutical compositions comprise Tris buffer of aboutpH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and may furtherinclude sorbitol or a suitable substitute therefor. In certainembodiments, the composition may be prepared for storage by mixing theselected composition having the desired degree of purity with optionalformulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in theform of a lyophilized cake or an aqueous solution. Further, in someembodiments, the antibody or fragment may be formulated as alyophilizate using appropriate excipients such as sucrose.

The pharmaceutical compositions of the invention can be selected forparenteral delivery. Alternatively, the compositions may be selected forinhalation or for delivery through the digestive tract, such as orally.Preparation of such pharmaceutically acceptable compositions is withinthe skill of the art. The formulation components are present preferablyin concentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be provided in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired antibody or fragment in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which the antibody or fragment is formulated as asterile, isotonic solution, properly preserved. In certain embodiments,implantable drug delivery devices may be used to introduce the desiredantibody or fragment.

In some or any embodiments, the pharmaceutical composition describedherein comprises a calcium salt, an acetate buffer, a polyol and asurfactant. Exemplary calcium salts include, but are not limited to,calcium acetate, calcium carbonate and calcium chloride. In someembodiments, the calcium salt is at a concentration of at least 0.5 mM,at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM or atleast 10 mM. In certain embodiments, the concentration of calcium saltis not greater than 11 mM, no greater than 12 mM, no greater than 13 mM,no greater than 14 mM, no greater than 15 mM, no greater than 16 mM, nogreater than 17 mM, no greater than 18 mM, no greater than 19 mM, nogreater than 20 mM, no greater than 21 mM, no greater than 22 mM, nogreater than 23 mM, no greater than 24 mM, or no greater than 25 mM. Anyrange featuring a combination of the foregoing endpoints iscontemplated, including but not limited to from about 0.5 mM to about 10mM, about 5 mM to about 10 mM, or about 5 mM to about 15 mM.

In some embodiments, the pharmaceutical composition comprises an acetatebuffer (e.g., sodium acetate) having a concentration ranging from about0.1 mM to about 1000 mM (1 M). In some embodiments, the concentration ofthe acetate buffer is at least 5 mM, at least 6 mM, at least 7 mM, atleast 8 mM, at least 9 mM, at least 10 mM, at least 15 mM, at least 60mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, atleast 200 mM, at least 500 mM, at least 700 mM, or at least 900 mM. Insome embodiments, the concentration of the acetate buffer is no greaterthan 10 mM, no greater than 15 mM, no greater than 20 mM, no greaterthan 25 mM, no greater than 30 mM, no greater than 35 mM, no greaterthan 40 mM, no greater than 45 mM, no greater than 50 mM, no greaterthan 55 mM, no greater than 60 mM, no greater than 65 mM, no greaterthan 70 mM, no greater than 75 mM, no greater than 80 mM, no greaterthan 85 mM, no greater than 90 mM, no greater than 95 mM or no greaterthan 100 mM. Any range featuring a combination of the foregoingendpoints is contemplated, including but not limited to from about 5 mMto about 15 mM, or from about 5 mM to about 10 mM or from about 10 mM toabout 25 mM. The buffer is preferably added to a concentration thatmaintains pH around 5-6 or 5-5.5 or 4.5-5.5. When the calcium salt inthe formulation is calcium acetate, in some embodiments, the totalconcentration of acetate is about 10 mM to about 55 mM, or about 20 mMto about 40 mM.

In some aspects, the pharmaceutical composition comprises a totalconcentration of acetate that is at least about 10 mM, at least about 15mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, atleast about 35 mM, at least about 40 mM, 45 mM, or 50 mM. In someembodiments, the concentration of acetate is no greater than about 30mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80mM, 85 mM, or 90 mM. Any range featuring a combination of the foregoingendpoints is contemplated, including but not limited to: about 10 mM toabout 50 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM,about 30 mM to about 50 mM, or about 30 mM to about 75 mM. In someembodiments, the calcium salt is calcium acetate and the acetate bufferis sodium acetate. By way of nonlimiting example, a solution containing10 mM calcium acetate will have 20 mM acetate anion and 10 mM of calciumcation, because of the divalent nature of the calcium cation, while asolution containing 10 mM sodium acetate will have 10 mM sodium cationand 10 mM acetate anion.

In some embodiments, the total concentration of ions (cations andanions) in solution is at least 10 mM, at least about 15 mM, at leastabout 20 mM, at least about 25 mM, at least about 30 mM, at least about35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM,at least about 55 mM, at least about 60 mM, at least about 65 mM, atleast about 70 mM, at least about 75 mM, at least about 80 mM, or atleast about 85 mM. In some embodiments, the total concentration of ionsis no greater than about 30 mM, no greater than about 35 mM, no greaterthan about 40 mM, no greater than about 45 mM, no greater than about 50mM, no greater than about 55 mM, no greater than about 60 mM, no greaterthan about 65 mM, no greater than about 70 mM, no greater than about 75mM, no greater than about 80 mM, no greater than about 85 mM, no greaterthan about 90 mM, no greater than about 95 mM, no greater than about 100mM, no greater than about 110 mM, no greater than about 120 mM, nogreater than about 130 mM, no greater than about 140 mM, no greater thanabout 150 mM, no greater than about 160 mM, no greater than about 170mM, no greater than about 180 mM, no greater than about 190 mM or nogreater than about 200 mM. Any range featuring a combination of theforegoing endpoints is contemplated, including but not limited to: about30 mM to about 60 mM, or about 30 mM to about 70 mM, or about 30 mM toabout 80 mM, or about 40 mM to about 150 mM, or about 50 mM to about 150mM. By way of nonlimiting example, a solution of 10 mM calcium acetatewill have a 30 mM total concentration of ions (10 mM cations and 20 mManions).

In some or any embodiments, the pharmaceutical composition comprises apolyol. Polyols encompass a class of excipients that includes sugars(e.g. mannitol, sucrose, sorbitol) and other polyhydric alcohols (e.g.,glycerol and propylene glycol). Exemplary polyols include, but are notlimited to, propylene glycol, glycerin (glycerol), threose, threitol,erythrose, erythritol, ribose, arabinose, arabitol, lyxose, maltitol,sorbitol, sorbose, glucose, mannose, mannitol, levulose, dextrose,maltose, trehalose, fructose, xylitol, inositol, galactose, xylose,fructose, sucrose, 1,2,6-hexanetriol and the like. Higher order sugarsinclude, but are not limited to, dextran, propylene glycol, orpolyethylene glycol. Reducing sugars such as fructose, maltose orgalactose oxidize more readily than do non-reducing sugars. Additionalexamples of sugar alcohols are glucitol, maltitol, lactitol oriso-maltulose. Additional exemplary lyoprotectants include glycerin andgelatin, and the sugars mellibiose, melezitose, raffinose, mannotriose,and stachyose. Examples of reducing sugars include glucose, maltose,lactose, maltulose, iso-maltulose and lactulose. Examples ofnon-reducing sugars include non-reducing glycosides of polyhydroxycompounds selected from sugar alcohols and other straight chainpolyalcohols. Monoglycosides include compounds obtained by reduction ofdisaccharides such as lactose, maltose, lactulose and maltulose.

In some or any embodiments, the pharmaceutical composition comprises apolyol at a concentration ranging from about 0% to about 40% w/v. Insome or any embodiments, the compositions comprise a polyol atconcentration of at least 0.5, at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 11, at least 12, at least 13, at least 14, at least15, at least 16, at least 17, at least 18, at least 19, at least 20, atleast 30, or at least 40% w/v. In some or any embodiments, thecomposition comprises a polyol at a concentration of about 1, 2, 3, 4,5, 6, 7, 8, 9% to about 10% w/v. In some or any embodiments, thecomposition comprises a polyol at a concentration of about 2% to about6% w/v. In some or any embodiments, the composition comprises a polyolat a concentration of about 4% w/v. In some or any embodiments, thecomposition comprises a polyol at about 6% w/v.

In some or any embodiments, the pharmaceutical composition comprises asurfactant. Exemplary surfactants include, but are not limited to,anionic, cationic, nonionic, zwitterionic, and amphoteric surfactantsincluding surfactants derived from naturally-occurring amino acids.Anionic surfactants include, but are not limited to, sodium laurylsulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate,chenodeoxycholic acid, N-lauroylsarcosine sodium salt, lithium dodecylsulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate,sodium deoxycholate, and glycodeoxycholic acid sodium salt. Cationicsurfactants include, but are not limited to, benzalkonium chloride orbenzethonium chloride, cetylpyridinium chloride monohydrate, andhexadecyltrimethylammonium bromide. Zwitterionic surfactants include,but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12. Non-ionicsurfactants include, but are not limited to, digitonin, Triton X-100,Triton X-114, TWEEN-20, and TWEEN-80. In another embodiment, surfactantsinclude, but are not limited to, lauromacrogol 400, polyoxyl 40stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50 and 60,glycerol monostearate, polysorbate 20, polysorbate 40, polysorbate 60,polysorbate 65 and polysorbate 80, soy lecithin and other phospholipidssuch as DOPC, DMPG, DMPC, and DOPG; sucrose fatty acid ester, methylcellulose and carboxymethyl cellulose. In some or any embodiments, thesurfactant is polysorbate 20.

Surfactants may be included in the compositions either individually oras a mixture in different ratios. In some or any embodiments, thecomposition comprises a surfactant at a concentration of about 0% toabout 5% w/v (e.g., about 0.001, about 0.002, about 0.005, about 0.007,about 0.01, about 0.05, about 0.1, about 0.2, about 0.3, about 0.4,about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, or about4.5% w/v). In some or any embodiments, the composition comprises asurfactant at a concentration of about 0.001% to about 0.5% w/v. In someor any embodiments, the composition comprises a surfactant at a aconcentration of about 0.004, about 0.005, about 0.007, about 0.01,about 0.05, or about 0.1% w/v to about 0.2% w/v. In some or anyembodiments, the composition comprises a surfactant at a concentrationof about 0.01% to about 0.1% w/v.

In some or any embodiments, the pharmaceutical composition comprises 55mM acetate, 13 mm calcium, 6.0% (w/v) sucrose, 0.006% (w/v) polysorbate20, pH 5.2.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving antigen binding proteins insustained- or controlled-delivery formulations. Techniques forformulating a variety of other sustained- or controlled-delivery means,such as liposome carriers, bio-erodible microparticles or porous beadsand depot injections, are also known to those skilled in the art. See,for example, International Patent Application No. PCT/US93/00829, whichis incorporated by reference and describes controlled release of porouspolymeric microparticles for delivery of pharmaceutical compositions.Sustained-release preparations may include semipermeable polymermatrices in the form of shaped articles, e.g., films, or microcapsules.Sustained release matrices may include polyesters, hydrogels,polylactides (as disclosed in U.S. Pat. No. 3,773,919 and EuropeanPatent Application Publication No. EP058481, each of which isincorporated by reference), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater.Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinylacetate (Langer et al., 1981, supra) or poly-D(−)-3-hydroxybutyric acid(European Patent Application Publication No. EP133988). Sustainedrelease compositions may also include liposomes that can be prepared byany of several methods known in the art. See, e.g., Eppstein et al.,1985, Proc. Natl. Acad. Sci. U.S.A. 82:3688-3692; European PatentApplication Publication Nos. EP036676; EP088046 and EP143949,incorporated by reference.

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method may beconducted either prior to or following lyophilization andreconstitution. Compositions for parenteral administration can be storedin lyophilized form or in a solution. Parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Free amino acids can be used in antibody or fragment formulations inaccordance with various embodiments of the invention as bulking agents,stabilizers, and antioxidants, as well as other standard uses. Lysine,proline, serine, and alanine can be used for stabilizing proteins in aformulation. Glycine is useful in lyophilization to ensure correct cakestructure and properties. Arginine may be useful to inhibit proteinaggregation, in both liquid and lyophilized formulations. Methionine isuseful as an antioxidant.

Embodiments of antibody formulations may further comprise one or moreantioxidants. To some extent deleterious oxidation of proteins can beprevented in pharmaceutical formulations by maintaining proper levels ofambient oxygen and temperature and by avoiding exposure to light.Antioxidant excipients can be used as well to prevent oxidativedegradation of proteins. Among useful antioxidants in this regard arereducing agents, oxygen/free-radical scavengers, and chelating agents.Antioxidants for use in therapeutic protein formulations in accordancewith the invention preferably are water-soluble and maintain theiractivity throughout the shelf life of a product. EDTA is a preferredantioxidant in accordance with the invention in this regard.

Formulations in accordance with the invention may include metal ionsthat are protein co-factors and that are necessary to form proteincoordination complexes, such as zinc necessary to form certain insulinsuspensions. Metal ions also can inhibit some processes that degradeproteins. However, metal ions also catalyze physical and chemicalprocesses that degrade proteins.

Magnesium ions (10-120 mM) can be used to inhibit isomerization ofaspartic acid to isoaspartic acid. Ca⁺² ions (up to 100 mM) can increasethe stability of human deoxyribonuclease. Mg⁺², Mn⁺², and Zn⁺², however,can destabilize rhDNase. Similarly, Ca⁺² and Sr⁺² can stabilize FactorVIII, it can be destabilized by Mg⁺², Mn⁺² and Zn⁺², Cu⁺² and Fe⁺², andits aggregation can be increased by Al⁺³ ions.

Embodiments of the antibody formulations can further comprise one ormore preservatives.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,crystal, or as a dehydrated or lyophilized powder. Such formulations maybe stored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration. The invention alsoprovides kits for producing a single-dose administration unit. The kitsof the invention may each contain both a first container having a driedprotein and a second container having an aqueous formulation. In certainembodiments of this invention, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are provided.

The therapeutically effective amount of an antibody-containingpharmaceutical composition to be employed will depend, for example, uponthe therapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment will varydepending, in part, upon the molecule delivered, the indication(s) forwhich the antibody is being used, the route of administration, and thesize (body weight, body surface or organ size) and/or condition (the ageand general health) of the patient.

Stability

The terms “stability” and “stable” as used herein in the context of acomposition comprising an antibody (or antigen binding fragment thereof)refer to the resistance of the antibody (or antigen binding fragmentthereof) in the composition to aggregation, degradation or fragmentationunder given manufacture, preparation, transportation and/or storageconditions. Antibody formulations comprising a high degree of stabilitydemonstrate enhanced reliability and safety and, as such, areadvantageous for clinical use.

Antibody stability in a composition is optionally assessed by examininga desired parameter of the antibody in the composition (e.g.,aggregation, degradation of heavy and/or light chains, chemicalmodification, etc.) over time. In this regard, a parameter is typicallyexamined at an initial time point (T0) and an assessment time point(T1), optionally while exposing the antibody to any of a number ofenvironmental conditions, and compared. An initial time point can be,for instance, the time that the antibody is first formulated in acomposition or first examined for quality (i.e., examined to determinewhether the antibody composition meets regulatory or manufacturingspecifications with respect to aggregation or degradation). An initialtime point also can be the time at which the antibody is reformulated ina composition (e.g., reformulated at a higher or lower concentrationcompared to an initial preparation). An assessment time point is, invarious embodiments, about 1 week (or about 2 weeks, or about 3 weeks,or about 4 weeks, or about 5 weeks, or about 6 weeks, or about 7 weeks,or about 8 weeks, or about 10 weeks, or about 3 months, or about 6months or about 1 year) after the initial time point. The desiredparameter (e.g., aggregation or degradation) of the antibody or fragmentthereof in the composition can be assessed under a variety of storageconditions, such as temperatures of −30° C., 4° C., 20° C. or 40° C.,shaking, pH, storage in different container materials (e.g., glassvials, pre-filled syringes, etc.), and the like.

Exemplary methods for determining the degree of aggregation, and/ortypes and/or sizes of aggregates present in a composition comprising theantibody include, but are not limited to, size exclusion chromatography(SEC), high performance size exclusion chromatography (HPSEC), staticlight scattering (SLS), Fourier Transform Infrared Spectroscopy (FTIR),circular dichroism (CD), urea-induced protein unfolding techniques,intrinsic tryptophan fluorescence, differential scanning calorimetry,and 1-anilino-8-naphthalenesulfonic acid (ANS) protein bindingtechniques. Size exclusion chromatography (SEC) may be performed toseparate molecules on the basis of their size, by passing the moleculesover a column packed with the appropriate resin, the larger molecules(e.g. aggregates) will elute before smaller molecules (e.g. monomers).The molecules are generally detected by UV absorbance at 280 nm and maybe collected for further characterization. High pressure liquidchromatographic columns are often utilized for SEC analysis (HP-SEC).Alternatively, analytical ultracentrifugation (AUC) may be utilized. AUCis an orthogonal technique which determines the sedimentationcoefficients of macromolecules in a liquid sample Like SEC, AUC iscapable of separating and detecting antibody fragments/aggregates frommonomers and is further able to provide information on molecular mass.Antibody aggregation in a composition may also be characterized byparticle counter analysis using a coulter counter or by turbiditymeasurements using a turbidimeter. Turbidity is a measure of the amountby which the particles in a solution scatter light and, thus, may beused as a general indicator of protein aggregation. In addition,non-reducing polyacrylamide gel electrophoresis (PAGE) or capillary gelelectrophoresis (CGE) may be used to characterize the aggregation and/orfragmentation state of antibodies or antibody fragments in acomposition.

Exemplary methods for determining antibody degradation include, but arenot limited to, size-exclusion chromatography (SEC), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and capillaryelectrophoresis with SDS (CE-SDS) and reversed phase HPLC with in-lineMS detection.

In various embodiments, less than 5% of the antibody described herein inthe composition is in aggregate form under conditions of interest. Forinstance, less than 4%, or less than 3%, or less than 2%, or less than1% of the antibody in the composition is in aggregate form after storageat −30° C., 4° C., 20° C. or 40° C. for a period of about 1 week (orabout 2 weeks, or about 3 weeks, or about 4 weeks, or about 5 weeks, orabout 6 weeks, or about 7 weeks, or about 8 weeks, or about 10 weeks, orabout 3 months, or about 6 months or about 1 year). In some embodiments,less than 5% (or less than 4% or less than 3% or less than 2% or lessthan 1% or less) of the antibody described herein in the composition isin aggregate form after storage for two weeks at about 4° C.

For example at least 85% (or at least 90%, or at least 91%, or at least92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%,or at least 97%, or at least 98%, or at least 99%) of antibody in acomposition optionally is present in non-aggregate (i.e., monomeric)form after storage at −30° C., 4° C., 20° C. or 40° C. for a period ofabout 1 week (or about 2 weeks, or about 3 weeks, or about 4 weeks, orabout 5 weeks, or about 6 weeks, or about 7 weeks, or about 8 weeks, orabout 10 weeks, or about 3 months, or about 6 months or about 1 year).In some embodiments, at least 85% (or at least 90%, or at least 91%, orat least 92%, or at least 93%, or at least 94%, or at least 95%, or atleast 96%, or at least 97%, or at least 98%, or at least 99% or more) ofthe antibody is present in the composition in non-aggregate form aftertwo weeks of storage at about 4° C. In some embodiments, at least 99% ofthe antibody is present in the composition in non-aggregate form afterstorage for two weeks at about 4° C. for two weeks and/or at least 95%of antibody present in the composition is in non-aggregate form afterstorage for two weeks at 40° C.

In various embodiments, less than 5% of the antibody described herein inthe composition is degraded. For instance, less than 4%, or less than3%, or less than 2%, or less than 1% or less of the antibody in thecomposition is degraded under conditions of interest. For example,optionally at least 85% (or at least 90%, or at least 91%, or at least92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%,or at least 97%, or at least 98%, or at least 99%) of the antibody isintact (i.e., not degraded) in a composition stored at about −30° C.,about 4° C., about 20° C. or about 40° C. for a period of about 1 week(or about 2 weeks, or about 3 weeks, or about 4 weeks, or about 5 weeks,or about 6 weeks, or about 7 weeks, or about 8 weeks, or about 10 weeks,or about 3 months, or about 6 months or about 1 year). In some aspects,at least 85% (or at least 90%, or at least 91%, or at least 92%, or atleast 93%, or at least 94%, or at least 95%, or at least 96%, or atleast 97%, or at least 98%, or at least 99% or more) of the antibody isintact (i.e., non-degraded) after storage in a composition at about 4°C. for a period of two weeks. In some embodiments, at least 99% of theantibody remains intact when stored in a composition at about 4° C. fortwo weeks and/or at least 95% remains intact when stored in acomposition at about 40° C. for two weeks.

Functional or activity stability of the antibody in a composition alsois contemplated herein. Assays for detecting and/or quantifying, e.g.,antibody binding to a target or sclerostin neutralization are known inthe art. Optionally, the antibody demonstrates about 50-100% activityunder conditions of interest compared to the activity of the antibody atthe initial time point. For example, the antibody retains a level ofactivity of between about 60-90% or 70-80% compared to the activity theinitial time point. Accordingly, functional stability of the antibodyincludes retention of activity of at least about 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or 100% and can include activitymeasurements greater than 100% such as 105%, 110%, 115%, 120%, 125% or150% or more compared to the activity at the initial time point.

Viscosity

In some embodiments, the viscosity of a composition comprising one ormore of the antibodies described herein is determined. The term“viscosity” as used herein refers to “absolute viscosity.” Absoluteviscosity, sometimes called dynamic or simple viscosity, is the productof kinematic viscosity and fluid density (Absolute Viscosity=KinematicViscosity×Density). The dimension of kinematic viscosity is L²/T where Lis a length and T is a time. Commonly, kinematic viscosity is expressedin centistokes (cSt). The SI unit of kinematic viscosity is mm²/s, whichis 1 cSt. Absolute viscosity is expressed in units of centipoise (cP).The SI unit of absolute viscosity is the millipascal-second (mPa-s),where 1 cP=1 mPa-s.

The viscosity of a composition can be measured hours (e.g., 1-23 hours),days (e.g., 1-10 days), weeks (e.g., 1-5 weeks), months (e.g., 1-12months), or years (e.g., 1-2 years, 1-3 years) after the addition of theantibody to the composition. Viscosity measurements may be made at astorage or administration temperature, e.g. 2-8° C. or 25° C. (roomtemperature). In some embodiments, absolute viscosity of the liquid orreconstituted liquid composition at the storage and/or administrationtemperature is 15 cP or less, or 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4cP or less. In some embodiments, absolute viscosity of the liquid orreconstituted liquid composition is 6 cP or less.

In some embodiments, the viscosity of the antibody composition ismeasured prior to and after the addition of antibody. Methods ofmeasuring viscosity are well known in the art and include, for example,using a capillary viscometer, or a cone-plate rheometer. Any method maybe used provided the same method is used to compare the test andreference formulations.

Therapeutic Methods

The antibody and pharmaceutical compositions described herein are usefulfor treating or preventing bone-related disorders, such as bone-relateddisorders associated with abnormal osteoblast or osteoclast activity. Insome embodiments, the antibody is administered to a subject sufferingfrom a bone related disorder selected from the group consisting ofachondroplasia, cleidocranial dysostosis, enchondromatosis, fibrousdysplasia, Gaucher's Disease, hypophosphatemic rickets, Marfan'ssyndrome, multiple hereditary exotoses, neurofibromatosis, osteogenesisimperfecta, osteopetrosis, osteopoikilosis, sclerotic lesions,pseudoarthrosis, pyogenic osteomyelitis, periodontal disease,anti-epileptic drug induced bone loss, primary and secondaryhyperparathyroidism, familial hyperparathyroidism syndromes,weightlessness induced bone loss, osteoporosis in men, postmenopausalbone loss, osteoarthritis, renal osteodystrophy, infiltrative disordersof bone, oral bone loss, osteonecrosis of the jaw, juvenile Paget'sdisease, melorheostosis, metabolic bone diseases, mastocytosis, sicklecell anemia/disease, organ transplant related bone loss, kidneytransplant related bone loss, systemic lupus erythematosus, ankylosingspondylitis, epilepsy, juvenile arthritides, thalassemia,mucopolysaccharidoses, Fabry Disease, Turner Syndrome, Down Syndrome,Klinefelter Syndrome, leprosy, Perthe's Disease, adolescent idiopathicscoliosis, infantile onset multi-system inflammatory disease, WinchesterSyndrome, Menkes Disease, Wilson's Disease, ischemic bone disease (suchas Legg-Calve-Perthes disease and regional migratory osteoporosis),anemic states, conditions caused by steroids, glucocorticoid-inducedbone loss, heparin-induced bone loss, bone marrow disorders, scurvy,malnutrition, calcium deficiency, osteoporosis, osteopenia, alcoholism,chronic liver disease, postmenopausal state, chronic inflammatoryconditions, rheumatoid arthritis, inflammatory bowel disease, ulcerativecolitis, inflammatory colitis, Crohn's disease, oligomenorrhea,amenorrhea, pregnancy-related bone loss, diabetes mellitus,hyperthyroidism, thyroid disorders, parathyroid disorders, Cushing'sdisease, acromegaly, hypogonadism, immobilization or disuse, reflexsympathetic dystrophy syndrome, regional osteoporosis, osteomalacia,bone loss associated with joint replacement, HIV associated bone loss,bone loss associated with loss of growth hormone, bone loss associatedwith cystic fibrosis, chemotherapy-associated bone loss, tumor-inducedbone loss, cancer-related bone loss, hormone ablative bone loss,multiple myeloma, drug-induced bone loss, anorexia nervosa,disease-associated facial bone loss, disease-associated cranial boneloss, disease-associated bone loss of the jaw, disease-associated boneloss of the skull, bone loss associated with aging, facial bone lossassociated with aging, cranial bone loss associated with aging, jaw boneloss associated with aging, skull bone loss associated with aging, andbone loss associated with space travel.

In some embodiments, the antibodies described herein are useful forimproving outcomes in orthopedic procedures, dental procedures, implantsurgery, joint replacement, bone grafting, bone cosmetic surgery andbone repair such as fracture healing, nonunion healing, delayed unionhealing and facial reconstruction. A composition comprising one or moreantibodies may be administered before, during and/or after theprocedure, replacement, graft, surgery or repair.

In some embodiments, the antibodies described herein are useful for thetreatment of any fracture comprising a gap between two segments of bone(e.g., a gap of at least about 1 mm between two segments of bone). Insome or any embodiments, the gap is at least about 2 mm, at least about3 mm, at least about 4 mm, at least about 5 mm, at least about 6 mm, atleast about 7 mm, at least about 8 mm, at least about 9 mm, or at leastabout 1 cm or more. In some or any embodiments, the gap is about 5 mm to1 cm, or up to 1 cm. The terms “bone gap defect” and “segmental skeletaldefect” are used synonymously herein and refer to a gap between twosegments of bone (e.g., a gap of at least 1 mm).

Exemplary bone gap defects include, but are not limited to, a comminutedfracture, a non-union fracture, a segmental skeletal defect, surgicallycreated bone defects, surgically treated bone defects, and bone defectscreated from traumatic injury to the bone or disease (including, but notlimited to, arthritis, tumor removal (resection) or infection removal).In some or any embodiments, the bone gap defect is produced by removalof infected sections of bone or the removal of cancer from the bone dueto bone cancers including, but not limited to, osteosarcoma, Ewing'ssarcoma, chondrosarcoma, malignant fibrous histiocytoma, fibrosarcoma,and chordoma. In some or any embodiments, the bone gap defect is adevelopmental deformity, e.g., due to a genetic defect.

In some or any embodiments, the bone gap defect is produced by removalof sections of bone containing a benign tumor. Exemplary benign bonetumors include, but are not limited to, osteoma, osteoid osteoma,osteoblastoma, osteochondroma, enchondroma, chonrdomyxoid fibroma,aneurysmal bone cyst, unicameral bone cyst, fibrous dysplasia of boneand giant cell tumor of the bone.

Administration of the antibody enhances or accelerates bone gap defecthealing, thereby “treating” the bone gap defect. “Enhancing” bonehealing means mediating a level of bone healing beyond (i.e., greaterthan) the level of bone healing experienced in subjects (e.g., mammals,such as humans) not administered the sclerostin inhibitor (i.e., controlsubjects). Bone healing is evidenced by, for example, bridging status,improved bone volume, improved bone mineral content and density withinthe fracture gap (i.e., formation of bridging bone), mature bone callus,improved bone strength (optionally accompanied by a medically-acceptablelevel of bone stiffness), or improved patient use of the affected area.By “improved” is meant an increase or decrease (as desired) in themeasured parameter. The increase can be a return, in whole or in part,of the measured parameter to baseline level (e.g., the level prior tothe bone gap defect), to values provided in normative databases used inthe art, or to the contralateral functional level (e.g., return, inwhole or in part, to the functional capabilities of, for example, thecontralateral limb). In some cases, the increase can be an improvementbeyond baseline level. If desired, the measured parameters in patientsadministered one or more doses of the antibody can be compared to thesame parameters in fracture patients (optionally age and gender matched)not administered the antibody to further analyze the efficacy of themethods described herein.

Formation of bridging bone, bone mineral content and bone density,and/or mature boney callus at the site of bone defect may be measuredusing radiography (e.g., radiographic absorptometry), single- and/ordual-energy X-ray absorptometry, quantitative computed tomography (QCT),ultrasonography, radiography (e.g., radiographic absorptometry), andmagnetic resonance imaging. In some embodiments, the antibody may beadministered at a dose and for a time period effective to increasebridging bone formation, formation of bony callus, or bone density (orvolume) at the defect site by at least about 5% (about 6%, about 7%,about 8%, or about 9%). In some embodiments, bridging bone formation,formation of bony callus, or bone density at the defect site isincreased by at least about 10% (e.g., at least about 10%, at leastabout 12%, at least about 15%, at least about 18%, at least about 20%,or at least about 22%). In other embodiments, bridging bone formation,formation of bony callus, or bone density at the defect site isincreased by the sclerostin inhibitor at least about 25% (e.g., at leastabout 26% or at least about 28%). In yet other embodiments, bridgingbone formation, formation of bony callus, or bone density at the defectsite is increased at least about 30% (e.g., at least about 32%, at leastabout 35%, at least about 38%, or at least about 40%) or at least about50% (e.g., at least about 60%, at least about 70%, at least about 80%,at least about 90%, or about 100%). The increase or re-establishment ofbridging bone formation can be determined at 1 week, 2 weeks, 3 weeks,or 4 weeks following the initial administration of antibody.Alternatively, the bone density level can be determined after thetreatment period ends (e.g., 1 week, 2 weeks, 3 weeks, or 4 weeks afterthe treatment period ends). In one aspect, the method reduces the amountof time required to establish a desired level of bone formation, bonevolume, bony callus, or bone density (e.g., any percent increase in boneformation, bone mineral density, bony callus, or bone volume describedherein) compared to age and gender-matched patients that do not receivethe antibody, thereby reducing recovery time for a subject. For example,in one embodiment, the antibody reduces the amount of time required toincrease bone density or volume at the defect site at least about 10%(e.g., at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, or at leastabout 50%).

The antibody need not cure the subject of the disorder or completelyprotect against the onset of a bone-related disorder to achieve abeneficial biological response. The antibody may be usedprophylactically, meaning to protect, in whole or in part, against abone-related disorder or symptom thereof. The antibody also may be usedtherapeutically to ameliorate, in whole or in part, a bone-relateddisorder or symptom thereof, or to protect, in whole or in part, againstfurther progression of a bone-related disorder or symptom thereof.Indeed, the materials and methods of the invention are particularlyuseful for increasing bone mineral density, and optionally maintainingthe increased bone mineral density over a period of time.

In some embodiments, one or more administrations of an antibodydescribed herein are carried out over a therapeutic period of, forexample, about 1 week to about 18 months (e.g., about 1 month to about12 months, about 1 month to about 9 months or about 1 month to about 6months or about 1 month to about 3 months). In some embodiments, asubject is administered one or more doses of a antibody described hereinover a therapeutic period of, for example about 1 month to about 12months (52 weeks) (e.g., about 2 months, about 3 months, about 4 months,about 5 months, about 6 months, about 7 months, about 8 months, about 9months, about 10 months, or about 11 months).

In addition, it may be advantageous to administer multiple doses of theantibody or space out the administration of doses, depending on thetherapeutic regimen selected for a particular subject. In someembodiments, the antibody or fragment thereof is administeredperiodically over a time period of one year (12 months, 52 weeks) orless (e.g., 9 months or less, 6 months or less, or 3 months or less). Inthis regard, the antibody or fragment thereof is administered to thehuman once every about 3 days, or about 7 days, or 2 weeks, or 3 weeks,or 4 weeks, or 5 weeks, or 6 weeks, or 7 weeks, or 8 weeks, or 9 weeks,or 10 weeks, or 11 weeks, or 12 weeks, or 13 weeks, or 14 weeks, or 15weeks, or 16 weeks, or 17 weeks, or 18 weeks, or 19 weeks, or 20 weeks,or 21 weeks, or 22 weeks, or 23 weeks, or 6 months, or 12 months.

In some embodiments, one or more doses of the antibody are administeredin an amount and for a time effective to increase bone mineral densityor treat a bone disorder associated with decreased bone mineral density.In various embodiments, one or more doses comprising from about 50milligrams to about 1,000 milligrams of the antibody are administeredper week to a subject (e.g., a human subject). For example, a dose ofantibody can comprise at least about 5 mg, 15 mg, 25 mg, 50 mg, about 60mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 120 mg,about 150 mg, about 200 mg, about 210 mg, about 240 mg, about 250 mg,about 280 mg, about 300 mg, about 350 mg, about 400 mg, about 420 mg,about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg,about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg,about 950 mg or up to about 1,000 mg of antibody. Ranges between any andall of these endpoints are also contemplated, e.g. about 50 mg to about80 mg, about 70 mg to about 140 mg, about 70 mg to about 270 mg, about75 mg to about 100 mg, about 100 mg to about 150 mg, about 140 mg toabout 210 mg, or about 150 mg to about 200 mg, or about 180 mg to about270 mg, or about 280 to about 410 mg. The dose is administered at anyinterval, such as multiple times a week (e.g., twice or three times perweek), once a week, once every two weeks, once every three weeks, oronce every four weeks. In some or any embodiments, a dose of antibodyranging from about 120 mg to about 210 mg is administered twice a week.In some or any embodiments, a dose of about 140 mg of the antibody isadministered twice a week. In various aspects, a dose of about 210 mg ofantibody is administered once a month.

In some embodiments, the one or more doses of antibody can comprisebetween about 0.1 to about 50 milligrams (e.g., between about 5 andabout 50 milligrams), or about 1 to about 100 milligrams, of antibodyper kilogram of body weight (mg/kg). For example, the dose of antibodymay comprise at least about 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, about 2mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 20 mg/kg,about 25 mg/kg, about 26 mg/kg, about 27 mg/kg, about 28 mg/kg, about 29mg/kg, about 30 mg/kg, about 31 mg/kg, about 32 mg/kg, about 33 mg/kg,about 34 mg/kg, about 35 mg/kg, about 36 mg/kg, about 37 mg/kg, about 38mg/kg, about 39 mg/kg, about 40 mg/kg, about 41 mg/kg, about 42 mg/kg,about 43 mg/kg, about 44 mg/kg, about 45 mg/kg, about 46 mg/kg, about 47mg/kg, about 48 mg/kg, or about 49 mg/kg, or about 50 mg/kg, about 55mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg,about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or up toabout 100 mg/kg. Ranges between any and all of these endpoints are alsocontemplated, e.g., about 1 mg/kg to about 3 mg/kg, about 1 mg/kg toabout 5 mg/kg, about 1 mg/kg to about 8 mg/kb, about 3 mg/kg to about 8mg·kg, about 1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 20 mg/kg,about 1 mg/kg to about 40 mg/kg, about 5 mg/kg to about 30 mg/kg, orabout 5 mg/kg to about 20 mg/kg.

Monitoring Therapy

Antibody-mediated increases in bone mineral content or bone density maybe measured using single- and dual-energy X-ray absorptometry,ultrasound, computed tomography, radiography, and magnetic resonanceimaging. The amount of bone mass may also be calculated from bodyweights or by using other methods (see Guinness-Hey, Metab. Bone Dis.Relat. Res., 5:177-181 (1984)). Animal models are used in the art fortesting the effect of the pharmaceutical compositions and methods on,for example, parameters of bone loss, bone resorption, bone formation,bone strength, or bone mineralization that mimic conditions of humandisease such as osteoporosis and osteopenia. Examples of such modelsinclude the ovariectomized rat model (Kalu, Bone and Mineral, 15:175-192(1991); Frost and Jee, Bone and Mineral, 18:227-236 (1992); and Jee andYao, J. Musculoskel. Neuron. Interact., 1:193-207 (2001)). The methodsfor measuring antibody activity described herein also may be used todetermine the efficacy of other sclerostin inhibitors.

In humans, bone mineral density can be determined clinically using dualx-ray absorptiometry (DXA) of, for example, the hip and spine. Othertechniques include quantitative computed tomography (QCT),ultrasonography, single-energy x-ray absorptiometry (SXA), andradiographic absorptiometry. Common central skeletal sites formeasurement include the spine and hip; peripheral sites include theforearm, finger, wrist and heel. Except for ultrasonography, theAmerican Medical Association notes that BMD techniques typically involvethe use of x-rays and are based on the principle that attenuation of theradiation depends on thickness and composition of the tissues in theradiation path. All techniques involve the comparison of results to anormative database.

Alternatively, a physiological response to one or more anti-sclerostinantibodies can be gauged by monitoring bone marker levels. Bone markersare products created during the bone remodeling process and are releasedby bone, osteoblasts, and/or osteoclasts. Fluctuations in boneresorption and/or bone formation “marker” levels imply changes in boneremodeling/modeling. The International Osteoporosis Foundation (IOF)recommends using bone markers to monitor bone density therapies (see,e.g., Delmas et al., Osteoporos Int., Suppl. 6:S2-17 (2000),incorporated herein by reference). Markers indicative of bone resorption(or osteoclast activity) include, for example, C-telopeptide (e.g.,C-terminal telopeptide of type 1 collagen (CTX) or serum cross-linkedC-telopeptide), N-telopeptide (N-terminal telopeptide of type 1 collagen(NTX)), deoxypyridinoline (DPD), pyridinoline, urinary hydroxyproline,galactosyl hydroxylysine, and tartrate-resistant acid phosphatase (e.g.,serum tartrate-resistant acid phosphatase isoform 5b). Boneformation/mineralization markers include, but are not limited to,bone-specific alkaline phosphatase (BSAP), peptides released from N- andC-terminal extension of type I procollagen (P1NP, PICP), and osteocalcin(OstCa). Several kits are commercially-available to detect and quantifymarkers in clinical samples, such as urine and blood.

Combination Therapy

Treatment of a pathology by combining two or more agents that target thesame pathogen or biochemical pathway or biological process sometimesresults in greater efficacy and diminished side effects relative to theuse of a therapeutically relevant dose of each agent alone. In somecases, the efficacy of the drug combination is additive (the efficacy ofthe combination is approximately equal to the sum of the effects of eachdrug alone), but in other cases the effect is synergistic (the efficacyof the combination is greater than the sum of the effects of each druggiven alone). As used herein, the term “combination therapy” means thattwo or more agents are delivered in a simultaneous manner, e.g.,concurrently, or wherein one of the agents is administered first,followed by the second agent, e.g., sequentially.

In some embodiments, the antibody is administered along with a standardof care therapeutic for the treatment of decreased bone mineral density(i.e., the antibody and standard of care therapeutic are part of thesame treatment plan). As used herein, the term “standard of care” refersto a treatment that is generally accepted by clinicians for a certaintype of patient diagnosed with a type of illness. In some embodiments,the antibody is administered along with a second bone-enhancing agentuseful for the treatment of decreased bone mineral density or bonedefect. In some embodiments, the bone-enhancing agent is selected fromthe group consisting of an anti-resorptive agent, a bone-forming agent(i.e., anabolic), an estrogen receptor modulator (including, but notlimited to, raloxifene, bazedoxifene and lasofoxifene) and a drug thathas an inhibitory effect on osteoclasts. In some embodiments, the secondbone-enhancing agent is selected from the group consisting of abisphosphonate (including, but not limited to, alendronate sodium(FOSAMAX®), risedronate, ibandronate sodium (BONIVA®) and zoledronicacid (RECLAST®)); an estrogen or estrogen analogue; an anti-RANK ligand(RANKL) inhibitor, such as an anti-RANKL antibody (e.g., denosumab,PROLIA®); vitamin D, or a vitamin D derivative or mimic thereof; acalcium source, a cathepsin-K (cat-K) inhibitor (e.g. odanacatib),Tibolone, calcitonin or a calcitriol; and hormone replacement therapy.In some embodiments, the second bone-enhancing agent includes, but isnot limited to, parathyroid hormone (PTH) or a peptide fragment thereof,PTH-related protein (PTHrp), bone morphogenetic protein, osteogenin,NaF, a PGE2 agonist, a statin, strontium ranelate, and a sclerostininhibitor (e.g., an anti-sclerostin antibody described in, for example,U.S. Pat. No. 7,592,429 or 7,872,106). In some embodiments, the secondbone-enhancing agent is Forteo® (Teriparatide), Preotact®, or Protelos®.In some embodiments, the second bone-enhaiving agent comprises a bonemorphogenetic protein (e.g., BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6,BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14 and/orBMP-15).

In some embodiments, the combination therapy employing an antibodydescribed herein may precede or follow administration of additionaltherapeutic(s) (e.g., second bone-enhancing agent) by intervals rangingfrom minutes to weeks to months. For example, separate modalities areadministered within about 24 hours of each other, e.g., within about6-12 hours of each other, or within about 1-2 hours of each other, orwithin about 10-30 minutes of each other. In some situations, it may bedesirable to extend the time period for treatment significantly, whereseveral days (2, 3, 4, 5, 6 or 7 days) to several weeks (1, 2, 3, 4, 5,6, 7 or 8 weeks) lapse between the respective administrations ofdifferent modalities. Repeated treatments with one or bothagents/therapies of the combination therapy is specificallycontemplated.

Maintenance Therapeutic Regimen

Also contemplated is the use of a second bone-enhancing agent and/orantibody described herein in a maintenance regimen to, e.g., prevent orslow the loss of bone mineral density. In this regard, a method or usedescribed herein optionally comprises administering one or more amountsof a second bone-enhancing agent effective to maintain bone mineraldensity for a maintenance period of about 1 week to about 5 years afterthe treatment period with the antibody has ended. For example, in someembodiments, a method or use described herein comprises theadministration of a second bone-enhancing agent to the subject for amaintenance period of about at least about 1 week, about 2 weeks, about3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks,about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12weeks, about 3 months, about 13 weeks, about 14 weeks, about 15 weeks,about 16 weeks, about 4 months, about 17 weeks, about 18 weeks, about 19weeks, about 20 weeks, about 5 months, about 21 weeks, about 22 weeks,about 23 weeks, about 24 weeks, about 6 months, about 25 weeks, about 26weeks, about 27 weeks, about 28 weeks, about 7 months, about 29 weeks,about 30 weeks, about 31 weeks or longer (e.g., about 8 months, about 9months, about 10 months, about 11 months, about 1 year, about 15 months,about 18 months, about 2 years, about 3 years, about 4 years, about 5years or longer (e.g., over the lifetime of the subject). In someembodiments, the maintenance period is about 6-12 weeks. In someembodiments, the maintenance period is about 4-12 weeks, or about 1-3months. In some embodiments, the maintenance period is about 12-20weeks, or about 3-5 months. In some embodiments, the maintenance periodis about 20-32 weeks, or about 5-8 months. In some embodiments, themaintenance period is about 24-36 weeks, or about 6-9 months. In someembodiments, the maintenance period is about 1 year, about 2 years,about 3 years, about 4 years, about 5 years or longer. “Maintaining”bone mineral density includes maintaining similar levels of bone mineraldensity parameters experienced in the subject that received the antibodytreatment.

Kits

A pharmaceutical composition comprising one or more antibodies describedherein may be placed within containers (e.g., vials or syringes), alongwith packaging material that provides instructions regarding the use ofsuch pharmaceutical compositions. Generally, such instructions willinclude a tangible expression describing the antibody concentration, aswell as within certain embodiments, relative amounts of excipientingredients or diluents (e.g., water, saline or PBS) that may benecessary to reconstitute the pharmaceutical composition.

EXAMPLES Example 1—Analysis of Romosozumab PARG C-Terminal Variant

Wild-type romosozumab and a romosozumab PARG C-terminal variant weredigested by Lys-C and analyzed by LC/MS peptide mapping. The UV profilesof these two constructs were compared side by side (FIG. 3). It wasdetermined that wild-type romosozumab and the romosozumab PARGC-terminal variant have a similar peak eluting at 37.7 minutes butwild-type romosozumab was determined to have a mass of 659.3 Da and theromosozumab PARG C-terminal variant was determined to have a mass of886.7 Da. The majority of lysine (K) variants of romosozumab (PGK) wasthought to be removed from the process. The presence of a significantamount of the amidated form of the romosozumab PARG C-terminal variant(828.6 Da peak) confirms that the amidation efficiency is sequencedependent when compared to the wild-type romosozumab PG sequence.

Next, the PARG C-terminal variant was then treated with carboxypeptidase(CP-B), analyzed by CEX-HPLC method and compared with the PARGC-terminal variant control which was not treated by the CP-B. There is asignificant shift post treatment for the peaks eluting at 17.5 mins and21 mins, but not for the peak at 24 mins (FIG. 4). It is contemplatedthat the 24 min peak is the doubly amidated form, which is protectedfrom proteolytic degradation.

Example 2—C-terminal Variant Enrichment

Purification or enrichment for different romosozumab species from acomposition comprising wild-type romosozumab and the romosozumab PARGC-terminal variant is achieved by Cation Exchange Chromatography (CEX)fractionation. CEX separates proteins based on differences in theirsurface charges. At a set pH, positively charged variants of wild-typeromosozumab are separated on a cation-exchange column (e.g., Dionex ProPac WCX-10 analytical column, 2.0 mm×250 mm) and eluted using a saltgradient (e.g., Mobile Phase A: 10:90 (v/v) ACN, 19 mM MES pH 6.2;Mobile Phase B: 10:90 (v/v) ACN, 19 mM MES, 250 mM NaCl, pH 6.2). Thedifferent C-terminal variants of romosozumab are charged differently andthe more positively charged variant elutes later in CEX. Thus, theelution order is: PG (wild-type), P-amide (amidated proline ofwild-type), PARG variant, and PAR-amide. The fraction collector can beprogrammed to collect CEX eluents containing different variants atdifferent elution times.

Example 3—Analysis of Romosozumab PARG C-terminal Variant Aggregation

Without being bound to any particular theory, it is contemplated thatbecause the PARG C-terminal variant is highly charged, such forms wouldrepel non-amidated forms in the compositions, thus reducing aggregationin the composition.

Romosozumab PARG C-terminal variant protein A pool was analyzed side byside with wild-type romosozumab protein A pool using SEC-HPLC, a sizeexclusion HPLC method that separates protein based on differences intheir hydrodynamic volume (Table 1).

TABLE 1 Molecule % HMW AMG785 ARG ProA pool 3.4% AMG785 WT ProA pool7.2%

The data demonstrated that romosozumab PARG C-terminal variant has lesshigh molecular weight species as compared to the wild-type romosozumab.

Example 4—Viscosity Analysis of Romosozumab PARG C-terminal Variant

Antibody solutions containing romosozumab PARG C-terminal variant orwild-type romosozumab are measured using a cone and plate. The solutionsare concentrated up to 120 mg/mL according to approximate volumedepletion, and final concentrations are determined (±10%) using theproteins absorbance at 280 nm (after dilution to end up within 0.1-1absorbance units (AU)) and a protein specific extinction coefficient.Viscosity analysis is performed on a Brookfield LV-DVIII cone and plateinstrument (Brookfield Engineering, Middleboro, Mass., USA) using aCP-40 spindle and sample cup or an ARES-G2 rheometer (TA Instruments,New Castle, Del., USA) using a TA Smart Swap 2 degree cone/platespindle. All measurements are performed at 25° C. and controlled by awater bath attached to the sample cup. Multiple viscosity measurementswere collected, manually within a defined torque range (10-90%) byincreasing the RPM of the spindle. Measurements are averaged in order toreport one viscosity value per sample to simplify the resultingcomparison chart.

Example 5—Solubility Analysis of Romosozumb PARG C-terminal Variant

To determine the impact of the amino acid variation of romosozumab PARGvariant as compared to the wild type romosozumab on solubility uponsubcutaneous (SC) injection, a dialysis solubility assay was performedon both wild type and PARG-C terminal variant romosozumab in parallel.This screen entails dialyzing a sample of the romosozumab PARGC-terminal variant and a sample of the wild-type romosozumab into asolution that simulates the pH and ionic strength of the SC space andmonitoring the solubility and physical stability of the antibody inthese conditions over a short time period. Samples were formulated at˜63 mg/mL in formulation buffer (pH 5.2). Then each sample was injectedinto a dialysis cassette and dialyzed into a PBS buffer to mimic the SCspace. Visual observations were made 24 hours after initial dialysis.Wild-type romosozumab typically shows precipitation after 24 hours.

The results show that both molecules precipitate in this analysis butthe PARG C-terminal variant precipitates less and at a slower rate. Thissuggests that the variant is more resistant to precipitation than wildtype, although the variant does not abolish precipitation completely.

Example 6—Diffusion Analysis of Romosozumab PARG C-terminal Variant

To determine the impact of the amino acid variation of romosozumab PARGC-terminal variant as compared to the wild type romosozumab on diffusionfrom the subcutaneous (SC) space, an assay was performed using Scissor(Pion Inc., Billerica, Mass.). This assay entails injecting the samples(the romosozumab PARG C-terminal variant or wild-type romosozumab) at˜70 mg/mL into a simulated SC space comprised of a collagen andhyaluronic acid matrix. The antibody is able to diffuse out of thismatrix through a dialysis membrane into a reservoir of carbonate bufferat pH 7.4. Time points were collected for up to 3 days and each timepoint was assayed for protein concentration by RP-HPLC. The proteinconcentration vs. time curves generated simulate the diffusion ratesfrom the SC space. In addition, precipitation in the SC matrix ismonitored with visual inspection.

Both the wild type and PARG C-terminal variant romosozumab were testedin the Scissor as described above. The results shown in FIG. 5 indicatethat wild type romosozumab diffuses from the simulated SC space at amuch lower rate and more wild type romosozumab is retained at thesimulated injection site than PARG C-terminal variant romosozumab.

Example 7—FcRn Binding

FcRn, the neonatal Fc receptor, is an MHC class I-like heterodimercomposed of a transmembrane a chain (homologous to MHC class-I likemolecules) and a β2 microglobulin light chain. FcRn binds to theinterface between C_(H)2 and C_(H)3 domains of IgG heavy chains in theFc region of the IgG molecule under mildly acidic conditions (˜pH 6) andreleases it at neutral pH (˜7.4). By this highly pH-dependentinteraction, FcRn mediates IgG homeostasis in human adults bymaintaining serum IgG levels.

A competitive binding assay, the AlphaScreen® binding assay(PerkinElmer, San Jose, Calif.), was used to assess the binding of theFc domain of wild-type romosozumab and romosozumab PARG C-terminalvariant to FcRn. The assay is a bead based amplified luminescentproximity homogeneous assay (“Alpha”) that detects bimolecularinteractions. The assay contains two bead types, an acceptor bead and adonor bead. The acceptor beads are coated with a hydrogel that containsthioxene derivatives, as well as nickel chelate which binds to thehistidine domain of histidine labeled FcRn (FcRn-His). The donor beadsare coated with a hydrogel that contains phthalocyanine, aphotosensitizer, and streptavidin, which binds to biotinylated CHOderived human Fc. When FcRn-His and the biotinylated human Fc bindtogether, they bring the acceptor and donor beads into close proximity.When laser light is applied to this complex, ambient oxygen is convertedto singlet oxygen by the donor bead. If the beads are in closeproximity, an energy transfer to the acceptor bead occurs, resulting inlight production (luminescence), which is measured in a plate readerequipped for AlphaScreen® signal detection.

When an antibody is present at sufficient concentrations to inhibit thebinding of FcRn-His to the biotinylated human Fc domain, a dosedependent decrease in emission at 570 nm is observed. The test samplebinding relative to the antibody reference standard is determined andreported as % relative binding and can be used to demonstrate theintegrity of the Fc domain of the antibody. It is contemplated thatcompositions having the PARG C-terminal variant will have a similar orbetter dose response curve than the wild type antibody.

The results are shown in FIG. 6. It was observed that both wild typeromosozumab and PARG C-terminal variant romosozumab bound FcRn similarlyand FcRn binding was not affected by the PARG mutation.

Example 8—FcγRIIa Binding

FcγRIIa is an activating Fc receptor expressed on monocytes, certaindendritic cells, neutrophils, B cells, platelets and NK cells. FcγRIIa(CD32a) is the most widely distributed FcγR with two extracellularIg-like domains and low binding affinity for monomeric IgG. There aretwo common allelic variants in humans that are known to exist forFcγRIIa, expressing either histidine or arginine at position 131 (131Hand 131R, respectively).

A competitive binding assay was developed to assess the binding ofwild-type romosozumab and romosozumab PARG C-terminal variant to FcγRIIa(131H). The FcγRIIa (131H) binding assay is a bead-based amplifiedluminescent proximity homogeneous assay (AlphaScreen® binding assay(PerkinElmer, San Jose, Calif.) that detects bimolecular interactions.The assay contains 2 bead types, an acceptor bead and a donor bead. Theacceptor beads contain the fluorophore europium chelate and are coatedwith a hydrogel that contains glutathione, which binds recombinant humanFcγRIIa (131H)-glutathione-S-transferase (FcγRIIa (131H)-GST). The donorbeads are coated with a hydrogel that contains phthalocyanine, aphotosensitizer, and streptavidin, which binds to biotinylated humanIgG1. When FcγRIIa (131H)-GST and the biotinylated human IgG1 bindtogether, they bring the acceptor and donor beads into proximity. When alaser is applied to this complex, ambient oxygen is converted to singletoxygen by the donor bead. When the acceptor and donor beads are near,the singlet oxygen diffuses within the acceptor beads resulting in lightproduction (luminescence), which is measured in a plate reader equippedfor luminescence signal detection.

When antibody is present at sufficient concentrations to inhibit thebinding of FcγRIIa (131H)-GST to the biotinylated human IgG1, adose-dependent decrease in emission at 570 nm is measured. The testsample binding relative to the antibody reference standard is determinedand reported as % relative binding and can be used to demonstrate theintegrity of the Fc domain of the antibody. The results are shown inFIG. 7. It was observed that the relative binding of PARG C-terminalvariant romosozumab to FcγRIIa (131H) was much higher than wild-typeromosozumab.

Example 9—Mouse Pharmacokinetic Study

To evaluate in vivo drug exposure and bioavailability, a single dosepharmacokinetic study in mice is performed. Romosozumab PARG C-terminalvariant is injected either intravenously (via tail vein) orsubcutaneously at a dose of 1 mg/kg. Using nine animals per group,staggered sampling permits collection of data at a large number of timepoints without exceeding the maximum volume of blood that can be drawnfrom an individual animal. At each time point, 0.05 ml of blood isdrawn. Animals 1 to 3 are sampled at 0.083, 24, 96 and 192 hourspost-dose. Animals 4-6 are sampled at 1, 48, 168 and 240 hours. Animals7-9 are sampled at 6, 72 and 192 hours. Serum is collected from thewhole blood sample and test article concentration is determined by abinding immunoassay such as an ELISA (Enzyme-Linked ImmunoSorbantAssay). Changes in test article concentration over time can be used tocalculate pharmacokinetic parameters via two compartment analysis.Parameters of interest include, but not limited to, area under theplasma concentration-time curve (AUC), half-life (t_(1/2)) and clearance(CL) for each dose group. Bioavailability can be determined as the ratioof AUC for the subcutaneous dose to the AUC for the intravenous dose.

What is claimed is:
 1. An antibody that specifically binds to sclerostinof SEQ ID NO: 1 and comprises a set of six CDRs set forth in SEQ ID NOs:2-7, wherein the antibody comprises a heavy chain comprising the aminoacid sequence Pro-Ala-Arg-Gly (SEQ ID NO: 8) at the C-terminus of theheavy chain.
 2. The antibody of claim 1, wherein the antibody comprisesa light chain variable region comprising an amino acid sequence setforth in SEQ ID NO: 9 and a heavy chain variable region comprising anamino acid sequence set forth in SEQ ID NO:
 10. 3. The antibody of claim1 or claim 2, wherein the C-terminus of the heavy chain is amidated. 4.The antibody of any one of claims 1-3, wherein the C-terminus of bothheavy chains comprises the amino acid sequence Pro-Ala-Arg-Gly (SEQ IDNO: 8).
 5. The antibody of claim 4, wherein the C-terminus of both heavychains is amidated.
 6. The antibody of any one of claims 1-5, comprisingthe light chain amino acid sequence set forth in SEQ ID NO: 12 and theheavy chain amino acid sequence set forth in SEQ ID NO:
 13. 7. Theantibody of claim 1 or claim 2, wherein the antibody comprises asequence of amino acids comprising Pro-Ala-Arg-Gly-Lys (SEQ ID NO: 11)at the C-terminus of a heavy chain.
 8. The antibody of claim 7,comprising the light chain amino acid sequence set forth in SEQ ID NO:12 and the heavy chain amino acid sequence set forth in SEQ ID NO: 14.9. A pharmaceutical composition comprising a population of antibodies ofany one of claims 1-8 and a pharmaceutically acceptable carrier.
 10. Apharmaceutical composition comprising a mixture of antibodies thatspecifically bind to sclerostin of SEQ ID NO: 1 and a pharmaceuticallyacceptable carrier; wherein about 3-5% of the antibodies in thecomposition are a population of antibodies of any one of claims 1-8. 11.The pharmaceutical composition of claim 9 or claim 10, wherein all orpart of the population of antibodies comprise a single heavy chaincomprising a C-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence.
 12. Thepharmaceutical composition of claim 9 or claim 10, wherein all or partof the population of antibodies comprise a heavy chain comprising aC-terminal Pro-Ala-Arg-Gly (SEQ ID NO: 8) sequence that is amidated. 13.The pharmaceutical composition of claim 12, wherein less than about 35%of the population of antibodies is singly amidated.
 14. Thepharmaceutical composition of claim 10, wherein all or part of thepopulation of antibodies comprise a C-terminal Pro-Ala-Arg-Gly (SEQ IDNO: 8) sequence in both heavy chains.
 15. The pharmaceutical compositionof claim 14, wherein all or part of the population of antibodiescomprising two heavy chains comprising a C-terminal Pro-Ala-Arg-Gly (SEQID NO: 8) sequence are amidated on both heavy chains.
 16. Thepharmaceutical composition of claim 15, wherein less than about 35% ofthe population of antibodies are amidated on both heavy chains.
 17. Thepharmaceutical composition of any one of claims 9-16, wherein less thanabout 35% of the population of antibodies comprise heavy chains that arenot amidated.
 18. The pharmaceutical composition of any one of claims9-17, wherein about 33% of the population of antibodies are notamidated, about 33% of the population of antibodies comprise oneamidated heavy chain, and about 33% of the population of antibodiescomprise two amidated heavy chains.
 19. The pharmaceutical compositionof any one of claims 9-18, further comprising a calcium salt, an acetatebuffer, a polyol and a surfactant.
 20. The pharmaceutical composition ofclaim 19, wherein the calcium salt comprises calcium acetate.
 21. Thepharmaceutical composition of claim 19, wherein the acetate buffercomprises sodium acetate.
 22. The pharmaceutical composition of claim19, wherein the polyol comprises sucrose.
 23. The pharmaceuticalcomposition of claim 19, wherein the surfactant comprises polysorbate20.
 24. The pharmaceutical composition of claim any one of claims 9-23,further comprising 55 mM acetate, 13 mm calcium, 6.0% (w/v) sucrose,0.006% (w/v) polysorbate 20, pH 5.2.
 25. A method of increasing bonemineral density in a subject in need thereof comprising administeringthe composition of any one of claims 9-24 to the subject in an amounteffective to increase bone mineral density in the subject.